L Putorti 1 , Srour 1 , L Loisel 1 , C Poulin 2 and B Brais

1 _{Laboratoire de neurogénétique de la motricité, Centre de Recherche du Centre} Hospitalier de l’Université de Montréal

2 _{Montreal’s Children Hospital of the McGill University Health Center}

Correspondence to : Bernard Brais, MD, Mphil, PhD, Laboratoire de neurogénétique de la motricité, M4211-L3, Hôpital Notre-Dame, Centre de Recherche du Centre Hospitalier de l’Université de Montréal, 1560 Sherbrooke Est, Montréal, Québec, Canada, H2L 4M1 Telephone : (514) 890-8000, # 25560

Fax : (514) 412-7525

Keywords : hereditary sensory neuropathy ; retinitis pigmentosa ; ataxia ; genome-wide scan ; homozygocity mapping ; identity by descent

Abbreviations : DRG = dorsal root ganglion ; FC = French Canadian ; LOD = logarithm of the odds ; PCARP = posterior column ataxia and retinitis pigmentosa ; SNP = single nucleotide polymorphism ; STR = short tandem repeat

49 Abstract

Hereditary sensory neuropathies are a clinically and genetically heterogeneous group of disorders. Posterior column ataxia associated with a retinitis pigmentosa (PCARP, OMIM 609033) is a rare autosomal recessive sensory neuropathy. The study of a Spanish and American family with PCARP has allowed mapping of the causative locus to a 8.3 cM region on chromosome 1q31-q32. We recruited a large French-Canadian (FC) family with four cases of PCARP. Genome-wide scanning linked this family to the previously described PCARP locus (LOD score of 3.0). Because all FC cases come from the small Paspébiac village of Gaspésie well known for its Basque settlers in the XVIIIe century, the possibility that the FC cases may share the same historical mutation with the Spanish family of Rom background was raised. Comparison of the carrier haplotype between the Spanish and FC families established that they shared a rare 60 SNP marker haplotype (rs4543871-rs3738800) which enabled us to reduce the previously reported candidate interval to a 203 kb interval which contains only four genes. The sequencing of exons and intron/exon boarders of ATF3, FAM71A, BATF3 and NSL1 did not uncover any mutations.

50 Introduction

Hereditary sensory neuropathies are a clinically and genetically heterogeneous group of disorders 1-4_{. Berciano and Polo characterized an early-onset recessive ataxia} with retinitis pigmentosa and sensory neuropathy in seven cases belonging to two Rom families from Cantabria in Northern Spain 5_{. Nee and Higgins characterized a large} American family of Dutch extraction with six cases affected by a posterior column ataxia and retinitis pigmentosa (PCARP, OMIM 609033) 6_{. Comparing the Spanish and the} American-Dutch phenotypes led to the conclusion that all cases were affected by the same condition and that it should be referred to as PCARP considering that ataxia was mostly secondary to the sensory neuropathy 7_{. Loveless and Higgins first linked the American-} Dutch family to chromosome 1q31-q32 (LOD score of 8.94) 8. They defined an 8.3 cM candidate interval flanked by microsatellites markers D1S2692 and D1S414 8_{. In} collaboration with Berciano, Loveless and Higgins linked the Spanish cases to the same locus (LOD score of 3.56, D1S2692-D1S549, 11.6 Mb) 9_{. They observed discordant} haplotypes between the two families, suggesting that two historically distinct mutations arose in the two populations 9_{. We uncovered a large family with an autosomal recessive} sensory neuropathy with retinitis pigmentosa originating from the French-Canadian (FC) village of Paspébiac. Situated in the Gaspésie region of North-Eastern Quebec, Canada, it is known for the presence of other recessive diseases with founder effects and for its founding by some Basque settlers in the XVIIIe century 10;11_{. In this paper, we show that} this family is linked to the PCARP locus and that they likely carry the same historical mutation as the Spanish cases, which allowed us to fine map the locus to a 203 kb candidate interval containing only four genes.

51 Subjects and methods

Clinical assessment

Cases and family members were recruited from the Montreal’s Children Hospital of the McGill University Health Center and form the neuromuscular clinic of the Centre de Réadapation Marie-Enfant of the CHU Hôpital Sainte-Justine. Cases underwent a detailed neurological examination by experienced neurologists (B. B., C. P. and M. S.). Geographical and genealogical data were collected by a research nurse (L. L.). This research project was approved by the institutional ethics committee of the Centre de Recherche du Centre Hospitalier de l’Université de Montréal. Informed consent was obtained from participating cases and family members. DNA from Spanish cases IV-2, IV- 10, IV-14, V-1 and V-3 were generously provided by Dr J. J. Higgins and Dr J. Berciano 9.

Genome-wide scan and fine mapping

A first microsatellite genome-wide scan was performed at deCODE genetics (Reykjavik, IC) on cases IV-5, V-4 and VI-2 and family members III-3, III-4, IV-1, IV-2, IV-3, IV-4, IV-7, IV-8, V-1, V-2, V-3, VI-1 and VI-3 belonging to a FC family. Genotypes were generated for 500 STR markers separated by an average of 8 cM. Another SNP genome- wide scan was performed on cases VI-2 belonging to the FC family and on case V-3 belonging to the Spanish family 9_{. Genotypes were generated for 620 000 SNP markers} separated by an average of 4.7 kb based on the genomic sequence of the candidate region (http://www.genome.ucsc.edu, Mar 2006 assembly) using the Human 610-Quad BeadChip (Illumina, San Diego, CA, USA) at the Genome Quebec Innovation Centre of the McGill University (Montreal, QC, CA).

Linkage analysis

Multipoint parametric linkage analysis was performed using Genehunter v.2.1 12_. Marker order and genetic distances were based on the deCODE genetic map (Reykjavik, IC) and UCSC physical map (http://genome.ucsc.edu, Mar 2006 assembly). Allele frequencies provided by deCODE genetics (Reykjavik, IC) and based on genotypes of 186

52 chromosomes from FC individuals not participating in this study were used. The PCARP phenotype was analyzed as an autonomic recessive trait with 100% penetrance and with an estimated disease gene frequency of 0.001. No phenocopies were incorporated into the analysis. The haplotypes were reconstructed in a single section using the maximum probability method of Genehunter v.2.1 12_.

Sequencing of candidate genes (ATF3, FAM71A, BATF3 and NSL1)

DNA was extracted from peripheral blood lymphocytes using the Gentra Puregene Blood Kit (Qiagen, Mississauga, ON, CA) or from saliva using the Oragene DNA Extraction Kit (DNA Genotek, Kanata, ON, CA). RNA was extracted from immortalized lymphoblasts using Trizol (Invitrogen, Carlsbad, CA, USA). The RNA samples were treated with the Super Script III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) to obtain the reverse transcriptase products. The non specific primers used for the RT-PCR were the OligodT and the Random Hexamers (Invitrogen, Carlsbad, CA, USA). The resulting RT products were used as a template for PCR amplification of the entire coding region of the genes RNA messenger transcripts with the Advantage 2 Polymerase Mix (Clontech, Mountain View, CA, USA). Mutation screening of the four candidates ATF3, FAM71A,

BATF3 and NSL1 genes was performed by genomic sequencing of the entire coding and a

minimum of 30 pb of intronic flanking sequences and coding sequencing of the cDNA sequences. PCR primers used to amplify the DNA were designed using the ExonPrimer tool (http://ihg2.helmholtz-muenchen.de/ihg/ExonPrimer.html) or the Primer3 v.0.4.0 tool (http://fokker.wi.mit.edu/primer3/input.htm). Large sequences were divided into overlapping fragments. Primers were synthesized by Alpha DNA (Montréal, QC, CA), IDT (Coralville, IA, USA) and Invitrogen (Carlsbad, CA, USA).Fragments were amplified using a standard amplification mix. The PCR and RT-PCR products and primer pairs were sent to the Genome Quebec Innovation Centre of the McGill University (Montréal, QC, CA) for forward and reverse sequencing. Sequences were aligned using EditSeq v.5.02 (DNASTAR, Madison, WI, USA) and SeqMan v.5.01 (DNASTAR, Madison, WI, USA) and analysed using SeqMan v.5.01 (DNASTAR, Madison, WI, USA) and Mutation Surveyor v.3.1 (PA, USA). Polymorphisms were confirmed by amplification and sequencing of the specific region for the entire cohort.

53 Results

We recruited a large FC family in which we identified four cases affected by a childhood-onset hereditary sensory neuropathy with retinitis pigmentosa (Fig. 1). The two males and two females were 16-51 years old (Tab. I). They presented orthopedic deformities, scoliosis, mild distal weakness of intrinsic hand and foot muscles, and delayed walking. Their perception of fine touch and vibration was decreased. Proprioception was absent in the feet. Deep tendon reflexes were absent. Walking became impossible in all cases. The upper limb ataxia was present but mild. Recurrent urinary tract infections and incontinence were other features. Our first literature search at the time of recruitment for a recessive sensory neuropathy associated with a retinitis pigmentosa did not uncover the publications on PCARP 5-9. Therefore, we completed a genome-wide scan using STR markers and linked our family to the PCARP locus with a LOD score of 3,0 on chromosome 1q32.2-q41 (Fig. 2). A recombinant present in case VI-2 delimited a candidate interval of 6.7 Mb between STR markers D1S2692 and D1S237 (Fig. 1). SNP homozygosity mapping further refined the candidate interval to 5.3 Mb between SNP markers rs643930 and rs1434889 on chromosome 1q32.2-q32.3 (Data not shown). Considering the FC family Basque ancestors, we contacted Dr Berciano to establish if the FC cases and the Spanish cases could share the same mutation by descent, which could allow further fine mapping of the mutated gene. As shown in table II, the FC case VI-2 and the Spanish case V-3 share a 203 kb haplotype between SNP markers rs4543871 and rs3738800 at the 1q32.3 locus (Tab. II). We did not observe the same haplotype in more then 80 unrelated phased FC chromosomes (Data not shown). Together, our results are highly suggestive that indeed the Spanish-Rom mutation was introduced by one or a few immigrants to the Gaspésie region of Quebec. This allows us to substantionally narrow the previously published interval from 11.6 Mb to 203 kb 9_.

This 203 kb candidate interval contains four known genes: ATF3, FAM71A, BATF3 and NSL1 (Fig. 3). Nnfl-17 synthetic lethal (NSL1) is a kinetochore complex component and is required for normal chromosome alignment and segregation and kinetochore formation during mitosis 13-16_{; family 71A (FAM71A) is a hypothetical protein without known} function; basic ATF-like 3 (BATF3) is a leucine zipper transcription factor and functions as a repressor when heterodimerizing with JUN 17;18_{; and activating transcription factor 3} (ATF3) functions as a transcriptional repressor (longer isoform) and a transcriptional activator (shorter isoform) 19-21_{. ATF3 is by far the most interesting candidate gene of the} four because it is highly expressed in the dorsal root ganglion (DRG) neurons 19-21_{. ATF3}

54 was shown to contribute to nerve regeneration by increasing the intrinsic growth state of injured neurons 19-21_{. We have sequenced the complementary and genomic coding DNA of} all of these genes and have found no mutations. However, we have found a rare polymorphism (rs11119988) in the ATF3 gene with a heterozygosity of 0.062±0.165 situated 154 pb before the second exon, which further supports that this region is shared between the FC and Spanish families. In the UCSC database, there are no individuals sharing the same homozygote genotype as the FC and Spanish cases.

55 Conclusion

Here, we report a FC family originating form Paspébiac in Gaspésie affected by PCARP (OMIM 609033). The FC, Spanish and American-Dutch families are clinically similar. The FC family is also linked to the PCARP locus on chromosome 1q32.2-q32.3 as are the Spanish and American-Dutch families. Interestingly, Paspébiac witnessed a series of settlements by Basque fisherman from France and Spain after the XVIIIe century 11_. While assembling our genealogical and geographical data, we noticed that many family members had Basque family names. In particular, one shared Basque ancestor originated from Bidart in France near the Spanish Basque region settled in Paspébiac in the XVIIIe century. Based on these observations, we decided to compare the SNP haplotypes of a FC case with a Spanish case for our locus. We discovered a rare haplotype, common for both cases, of 203 kb spanning four genes as potential disease causing. Therefore, it appears that the FC and the Spanish families are distantly related. In the candidate interval, no mutation was found to date by classical exon and exon/intron sequencing. The

ATF3 gene remains the most intriguing candidate because of its role in transcriptional

activation and its high expression in DRG neurons 19-21_{. A search for rare alternatively} splices retinal DRG transcripts is underway as is full genomic sequencing of the ATF3 gene. This study illustrates the benefits of comparing European and FC samples in rare diseases and performing transatlantic identity by descent homozygosity fine-mapping to substantially narrow a candidate interval.

56 Acknowledgements

We would like to thank all family members for their participation at our study. This work was supported by the Fondation de la névrite sensitive. We would like to thank Dr J. J. Higgins and Dr J. Berciano for sharing with us some DNA on the Spanish PCARP cases.

57

Figure 1. Pedigree of the FC family with PCARP. The circles represent females and the squares represent males. The black circles and squares represent affected individuals with PCARP (n = 4). Symbols of unaffected individuals are not shaded. Reconstructed haplotypes for the five chromosome 1q markers, D1S2685, D1S2692, D1S245, D1S205 and D1S237 are shown below individuals. Haplotypes segregating with the disease are represented by boxed and black shaded symbols.

58 Table I. Clinical summary of two PCARP Paspebiac cases.

Case V-4 Case VI-2

Date of birth 20-06-1993 14-12-1994

Age in 2009 16 y 15 y

Age at exam 14 y 13 y

Age at diagnosis 18 mo 9 mo

Presentation Osteomyelitis Mutilation

Neonatal Seizures ---

Walked 4 y 2 y

Stopped walking --- 8 y

Scoliosis Yes Yes

Retinitis Pigmentosa Yes Yes

Recurrent Osteomyelitis Yes No

Recurrent UTI Yes Yes

Urinary incontinence Yes Yes

Fecal incontinence No Occasional

Orthostatic hypotension No No

Orthopedic deformities No major except scoliosis and finger contractures

Knees and legs

Hearing Normal Normal

Cognition Normal Normal

Charcot joints No Yes

Weakness Mild distal (intrinsic hand and foot) Mild distal (intrinsic hand and foot)

Reflexes Absent Absent

Vibration Absent Absent

Proprioception Moderate loss Mild loss

Fine touch Major loss Major loss

Nerve Biopsy Axonal loss, no onion bulbs, no abnormal inclusions

59

Figure 2. Multipoint LOD score distribution for the FC family PCARP. Data are shown for the nine chromosome 1q markers, D1S2818, D1S413, D1S2717, D1S249, D1S2685, D1S245, D1S205, D1S237 and D1S2641 at the 1q32.2-q41 locus.

60 Table II. Haplotypes for the FC case VI-2 and the Spanish case V-3 of PCARP on chromosome 1q32.3. Genotypes are shown for the 60 shared SNP in black. Carrier frequencies were taken from NCBI (http://www.ncbi.nlm.nih.gov/).

Marker Position VI-2 V-3 Frequency Marker Position VI-2 V-3 Frequency

rs4543871 210824281 GG AA --- rs3923950 210878498 GG GG 0.74 rs1877474 210824514 GG GG 0.39 rs3123537 210878570 AA AA 0.60 rs1105899 210825227 GG GG 0.87 rs3890801 210880126 GG GG 0.89 rs12401671 210825710 GG GG 0.88 rs3122718 210884070 CC CC 1.00 rs17019384 210827184 GG GG 0.93 rs1553626 210893916 GG GG 0.11 rs12739624 210827680 GG GG 0.83 rs2501841 210894464 CC CC 0.82 rs11119982 210831467 GG GG 0.39 rs2501842 210895371 AA AA 0.57 rs17019421 210832706 AA AA 1.00 rs2456821 210908485 AA AA 0.83 rs6682064 210834463 GG GG 0.13 rs2501846 210908557 GG GG 0.19 rs11119983 210834951 CC CC 0.13 rs2492789 210911657 AA AA 0.79 rs1567710 210835051 GG GG 0.77 rs2501849 210913232 AA AA 0.48 rs6670396 210835520 GG GG 0.87 rs12754070 210923344 GG GG 0.80 rs9430097 210838504 CC CC 0.20 rs2501857 210923955 GG GG 0.80 rs17019438 210839560 AA AA 0.71 rs906363 210925420 AA AA 0.88 rs3795837 210839951 AA AA 0.92 rs2221593 210940054 GG GG 0.87 rs17019442 210840963 AA AA 0.84 rs9658807 210943525 AA AA 0.88 rs12564392 210842152 CC CC 0.97 rs2244935 210967110 GG GG 0.94 rs3125296 210844909 AA AA 0.18 rs12747251 210969712 GG GG 0.51 rs4951629 210853506 AA AA 0.95 rs15702 210978459 AA AA 0.75 rs11571536 210853543 GG GG 0.92 rs2174780 210986030 GG GG 0.10 rs11571537 210853648 AA AA 0.97 rs1507358 210991984 AA AA 0.27 rs10475 210860236 GG GG 0.82 rs6540762 210992480 GG GG 0.25 rs1126700 210860472 AA AA 0.92 rs12404885 210993790 AA AA 0.27 rs11119989 210862315 GG GG 0.98 rs11120012 210995829 AA AA 0.52 rs7533306 210864513 GG GG 0.79 rs4951638 210997838 AA AA 0.62 rs3122712 210865600 GG GG 0.28 rs11808846 211010204 AA AA 0.73 rs3122713 210866493 GG GG 0.87 rs10863997 211014143 GG GG 0.22 rs3795842 210866572 GG GG 0.28 rs1007036 211017987 AA AA 0.25 rs17019491 210871588 GG GG 0.10 rs3738803 211022542 AA AA 0.78 rs3123536 210876229 AA AA 0.87 rs3738800 211027489 AA GG ---

61

62 Supplementary material

Table 1. Primers used for candidate genes amplification of the gDNA. Genes are ordered according to their centromeric-telomeric position.

Fragment Forward primer Reverse primer

ATF3_promalternatif CCGAACTTGCATCACCAGT CGGCGGACTGGTTACTTAGA ATF3_exon1 CAGGATGATGGAAGGCTGTC GAACTGCTCACTTTCCCACC ATF3_exon2 GGTGTTGGAGGTCTGGTGG AGAACATGCCCAACACACAG ATF3_exon3 GCTAGCATTGCCCTTGTCTG GGGAAACTATAACCAAGTAGGGG ATF3_exon4_1 CCAGGTACACCCCTGCATC CTGCCTGAATCCTAACGGTG ATF3_exon4_2 TTTAGGCCTTAACACACTGGC TGGAGAGTCCTAACCCTTTGG

ATF3_exon4_3 AACACAAAATCCATGGGCAG ATAATGGGGAGGGAAAGGC ATF3_exonalternatif GGCAACACGGAGTAAACGAC CGAGCACCGAGGACTCAC ATF3_3’UTRsupp CAGAATCGACTAAGCCACCA TCTGAGCCTTCAGTTCAGCA FAM71A_exon1_1 AACAAAGGGGTATGTGACAGC GGGCTGGTACGTGCAATG

FAM71A_exon1_2 GCACCGATATTTGAGAGCG CTGTAGATGTTTTGGGGATGG FAM71A_exon1_3 ACCTCTGCTGCTTATGCTGG GACCTGCTGGAGGACTTTTG FAM71A_exon1_4 AAGAAGGGAAAAGGACAGGG TGCAGCACAGTTGACATCAC BATF3_exon1 AAAAACACCGACCCCAAAAC GCTCAGGAGCCATTCCAG BATF3_exon2 CTGTTGTGAGCCCATTGGTA AACACTGGGCTGGAAGGAC BATF3_exon3 TCATGGGCAAGAGGTGAAC GTGGCTGTGGGAGAGTGG NSL1_exon1 GTTTCAGCTGCAGCGTCC GCGTGGGATCTTTCTGAATC NSL1_exon2 TTTCCTCTGTCACAAATCTGG ACTCCCCTAGTAAACAACTTCTTG NSL1_exon3 CTTGCCATATTTGTTTTCCTTC AACAAAATGTCACTTATCTCCACG

NSL1_exon4 TTCCTCAAAAGAATGGTACTGACTC GGGAGAATATCCTTGATGCTG

NSL1_exon5 TTAAGCTGAGGTGAATCGTCC GATTTATTTAAAGGACCGCTGAG NSL1_exon6_1 GGGCAACAAAGCAAGACTTC TTTTCTCAGACTTCTCCCAGTG NSL1_exon6_2 AACATAATTTGACTTGGAACTAATGG CTGCTATTGGTCATGCTTTTC NSL1_exon6_3 GGCAGACAGCTGAGATTTG GGCCAGGGAAAGAGGTC NSL1_exon6_4 AGGCACATGGGAGTTAAGGC CATATACAGCCATGTGAGCACC NSL1_exon6_5 ACTAAGCCATGGTGGTTCCC CAACACAGCAAGACCTTGTTTC NSL1_exon6_6 CACTGCCTAAGGCTGGAAAG GCGATGGCGTGATCTTG

NSL1_exon6_7 GAGGCTGGGAATTGGAGAC CAGGTGCAGTGGCTCATTC

NSL1_exon6_8 GTTTCAAAACAGGCGTGTGC CAAGTCTCAGGTATTTATGGACTG

NSL1_exon6_9 TCATGTAGCTCTGTGGCCC GGCTCCTAGCCTCTCCC

NSL1_exon6_10 GCAATTTGCCGTCAGTTAGG TGATCATGGCATGTAGTAAGCAC NSL1_exon6_11 TCCACTTTATGCCTGGAATTG GCCCCTAGAGCTAGTCCACC NSL1_exon6_12 CAAAGGGTTGTGAAACAGCTC CAAGTGGAGGAAGATAGTCCTAAG

NSL1_exonalt CTCTTCCGGAGGCTGAGG CGATCATATCACTGGCAAACA

NSL1_reprise1 ATTCAGTGGGAGGGCTGG GACCAGCCACATAGCGAAAC

NSL1_reprise2 TTACTGCAACCTCTGCCC TCGAGTGTGCTGTGTTTTGG

NSL1_reprise3 AAAATTACCTCCAAGGCATCC CATCCTTTAGCCTCCCAGTG NSL1_repriseinterne1 ACAAGGTCTTGCTGTGTTGC TCCCATACCCATTAGCAGTCA

63 Table 2. Primers used for candidate genes amplification of the cDNA. Genes are ordered according to their centromeric-telomeric position

Fragment Amorce forward Amorce reverse

SYT14_ADNc1 CGCATCATGGCGATTGAA TTTACCCAGCGCTTCATCTT SYT14_ADNc2 TTGATGCTGCTCCTTTTTCTC TGAGCATCTTGGACTGTTGC SYT14_ADNc3 ATTGCAGCCACCACCATATC GGATGCTGGTTTTTGCTCTC SYT14_ADNc4 AAGCTTCTGGTAACAGTGACAGC CTCTGCGGATGGATGTCTTG SYT14_ADNc5 AAAACACTTGATAGGTGGACAGG AAAGCATTTTGGTAGAAGGTTGA ATF3_ADNc1 CCAACCATGCCTTGAGGATA GCTGCTTCTCGTTCTTGAGC ATF3_ADNc2 GAAAAAGAGGCGACGAGAAA CTGGTCCAAGACCCACTCTG ATF3_ADNc3 AAGAGGCGACGAGAAAGAAA GTTCTCTGCTGCTGGGATTC ATF3_ADNc4 TGGGAGGACTCCAGAAGATG CTAACGGTGGGCATCCTG ATF3_ADNc5 CAGCAGCAGAGAACCATCAA GCACACACAATTTCTGGACA ATF3_ADNc6 CAGGCAGCAGTGTCTGTACC AGGACCTGCCATCATACTGC ATF3_ADNc7 GGCAATGTACTCTTCCGATG TGGTTTCCAAACATCTCTCCA ATF3_ADNc8 ATGATGGCAGGTCCTCTGTT TGAGAATTTCACAGAAACATCAGAA ATF3_ADNc9 GTGGTACCCAGGCTTTAGCA CCATGTCTTGGTTCCAATATTTA FAM71A_ADNc1 GAGAGGGTCGTCACTTCCTG CACTGGTGAGGCAGGTCTCT FAM71A_ADNc2 ACAGCAATCATCCGTGCTC TCTCCCCTTTTGGTGATCTG FAM71A_ADNc3 TGTGCTGAAAGACAACCATGA GTACAAACCTCAGGGGCAGA FAM71A_ADNc4 ATTGATGTGCACAACCGTGT GCTGGAATGCCACAGGTACT FAM71A_ADNc5 AACAGCTGCGCCTGAAGTT AGATGTTTTGGGGATGGATG FAM71A_ADNc6 CCGTGAACCTTCAAGGAAAG GTTGCCTTTGTTTCCTCCTG FAM71A_ADNc7 CATCCATCCCCAAAACATCT TGCATCCATGTCTGCTTCTG FAM71A_ADNc8 CTCCAAACAGCACGAAGGTG TTTTGGGCAATCTTGTCTCC FAM71A_ADNc9 AGAGCAAGAGCAGCCTGAGT TCCTCAGGAAAGAGCTGACC FAM71A_ADNc10 AGGTCCTCATTCAGCCACAG GGGCTTCAAAGGTCACTGTC

FAM71A_ADNc11 AGGAACGTCAGAGCCAACCT TGATTCTTTATTTAAACGGCAGTA BATF3_ADNc1 CAGACGTGGGACGGGAAG AGCCTTCTGGGTCTGCTTCT BATF3_ADNc2 GAGAGCGGGAAGCCTGAG GAGGCACTGGCACAAAGTT BATF3_ADNc3 ATGAGAGCCTGGAGCAAGAA GCACAAGGGCTCTGTGAGTT BATF3_ADNc4 ACACTCCTCTGCCCAGCA CAGGAAAAGGAAGTGTATTGTGC NSL1_ADNc1 CCCACAGTTCCGACGAAAA TGATTTCATCAAACTGATCTTCAA

NSL1_ADNc2 GCTCTGCCGGAGGAGATT GTTTCCCCTCTGCATTTCAA

NSL1_ADNc3 CCCAGAAAGATCCTGGAATG CAGAGGTTTTCCTGGAAGCA NSL1_ADNc4 TTGAAATGCAGAGGGGAAAC ATCATATCCCCGCACAGAGA

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66 3.3 Conclusion

L’histoire de Paspébiac nous apprend qu’il y a eu plusieurs vagues d’immigration de pêcheurs basques qui s’y sont établis. L’ancêtre commun le plus rapproché de nos cas canadiens-français possède un nom d’origine basque. Voilà pourquoi nous avons émis l’hypothèse que les cas de la famille canadienne-française pourraient porter la même mutation que les cas de la famille espagnole. Afin de valider notre hypothèse, nous avons